Accumulating evidence suggests that opioid tolerance rapidly develops in neonates and infants. The broad long-term objective of the proposed research is to understand underlying mechanisms of tolerance in the developing brain that would lead to improved pain treatment. The ventrolateral periaqueductal gray (vlPAG) is a key component of supraspinal pain-modulatory pathways, and plasticity in this area is strongly implicated in analgesic tolerance. This proposal focuses on understanding how adaptations in the vlPAG that occur with tolerance may differ between young, intermediate-aged and adult rats. In the first aim, the appearance of an immediate early gene, Fos, will be used as a marker of neuronal activation. Changes in the appearance of Fos with age and drug treatment will be correlated with behavioral measures of tolerance. The results will provide a basis for understanding how supraspinal mechanisms differentially contribute to morphine tolerance with age, and provide a tool for neurochemical identification of relevant cell populations. In the second aim, the involvement of the neuronal isoform of nitric oxide synthase (nNOS) in tolerance will be investigated. Although activation of nNos has been strongly implicated in tolerance, it's role in the vlPAG within different age groups remains poorly understood. The results of both aims will provide a foundation for long-term research into age- dependent mechanisms of tolerance. It is an important area of research that will provide novel insights into molecular mechanisms of opioid tolerance and will lead to new clinical interventions to improve treatment of pain, opioid tolerance and dependence that differ with age. PUBLIC HEALTH RELEVANCE: Although it has became apparent that neonates, infants, and children rapidly escalate opioid dose and develop tolerance faster, opioids remain mainstream therapy for perioperative, cancer and other chronic pain for pediatric populations. Therefore, opioid tolerance is a timely topic of great relevance to daily clinical practice of pediatric medicine and pediatric anesthesia. Using a rat model, this project examines neuronal circuits mediating opioid effects to identify differences between the adult and the developing brain mechanisms.